Genome-wide analyses of long noncoding RNA expression profiles correlated with radioresistance in nasopharyngeal carcinoma via next-generation deep sequencing

Abstract

Background: Radioresistance is one of the major factors limiting the therapeutic efficacy and prognosis of patients with nasopharyngeal carcinoma (NPC). Accumulating evidence has suggested that aberrant expression of long noncoding RNAs (lncRNAs) contributes to cancer progression. Therefore, here we identified lncRNAs associated with radioresistance in NPC.

Methods: The differential expression profiles of lncRNAs associated with NPC radioresistance were constructed by next-generation deep sequencing by comparing radioresistant NPC cells with their parental cells. LncRNA-related mRNAs were predicted and analyzed using bioinformatics algorithms compared with the mRNA profiles related to radioresistance obtained in our previous study. Several lncRNAs and associated mRNAs were validated in established NPC radioresistant cell models and NPC tissues.

Results: By comparison between radioresistant CNE-2-Rs and parental CNE-2 cells by next-generation deep sequencing, a total of 781 known lncRNAs and 2054 novel lncRNAs were annotated. The top five upregulated and downregulated known/novel lncRNAs were detected using quantitative real-time reverse transcription-polymerase chain reaction, and 7/10 known lncRNAs and 3/10 novel lncRNAs were demonstrated to have significant differential expression trends that were the same as those predicted by deep sequencing. From the prediction process, 13 pairs of lncRNAs and their associated genes were acquired, and the prediction trends of three pairs were validated in both radioresistant CNE-2-Rs and 6-10B-Rs cell lines, including lncRNA n373932 and SLITRK5, n409627 and PRSS12, and n386034 and RIMKLB. LncRNA n373932 and its related SLITRK5 showed dramatic expression changes in post-irradiation radioresistant cells and a negative expression correlation in NPC tissues (R = -0.595, p < 0.05).

Conclusions: Our study provides an overview of the expression profiles of radioresistant lncRNAs and potentially related mRNAs, which will facilitate future investigations into the function of lncRNAs in NPC radioresistance.

Keywords: Deep sequencing; Long noncoding RNA; Nasopharyngeal carcinoma; Radioresistance.

Fig. 1

The work flow of constructing long noncoding RNA profiles correlated with NPC radioresistance

Fig. 2

Overview of known lncRNAs associated with NPC radioresistance. a The length distributions of the transcripts. b The known lncRNAs identified in CNE-2 and CNE-2-Rs cells. The shaded area represented the lncRNAs found in both samples, and the areas coloured red and blue showed the number of lncRNAs expressed in samples CNE-2 and CNE-2-Rs, respectively. c Scatter plot of lncRNA expression profiles in radiosensitive CNE-2 (x-axis) and radioresistant CNE-2-Rs cells (y-axis). The significantly up-regulated lncRNAs were marked in red and the down-regulated lncRNAs in blue. |log2 Fold change| ≥ 1 and FDR < 0.001. d Validation of the expression levels of the top five up-regulated and down-regulated known lncRNAs via qRT-PCR

Fig. 3

Overview of novel lncRNA candidates associated with NPC radioresistance. a The length distributions of the transcripts. b The novel transcripts identified in CNE-2 and CNE-2-Rs cells. The shaded areas represented the lncRNAs found in both samples, and the areas coloured red and blue showed the number of lncRNAs expressed in samples CNE-2 and CNE-2-Rs, respectively. c Scatter plot of lncRNA expression profiles in radiosensitive CNE-2 (x-axis) and radioresistant CNE-2-Rs cells (y-axis). The significantly up-regulated novel lncRNAs were marked in red and the down-regulated lncRNAs in blue. |log2 Fold change| ≥ 1 and FDR < 0.001. d Validation of the expression of the top five up-regulated and down-regulated novel transcripts via qRT-PCR

Fig. 4

Validation of the lncRNAs and associated genes by qRT-PCR. Three pairs of lncRNAs and their associated genes exhibited corresponding changes in the prediction and both radioresistant cell lines, including lncRNA n373932 and SLITRK5, n409627 and PRSS12, and n386034 and RIMKLB

Fig. 5

Detection of the lncRNAs and associated genes by qRT-PCR in irradiated cells and NPC cells. a, b lncRNA n373932 and SLITRK5 had dramatic mRNA expression change in post-irradiation cell, expecially in radioresistant cells. c After making napierian logarithmic transformation and Bivariate Correlation analyses, we found that n373932 and SLITRK5 had a negative exprssion correlation (R = −0.595, p < 0.001, Fig. 5c)